There are many situations such as medical, veterinary and industrial applications, where mutant gene products are desirable. Microorganisms can produce a great variety of proteins, particularly enzymes, which can be used in may situations. Many of these proteins, however, have characteristics that are not ideal for subsequent use and therefore it is desirable to obtain mutants with altered activity or functions. There are a number of methods and techniques presently in use which can generate mutant genes and corresponding mutant gene products.
Stemmer (Stemmer, W. P. C. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389-391, 1994; Stemmer, W. P. C. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. USA 91; 10747-10751, 1994) has discussed the most effective methods to search sequence space in vitro to yield the greatest diversity of protein variants. Until recently, the most popular methods of creating combinatorial libraries are recursive strategies that seek to evolve sequences by the addition of point mutations (Cadwell and Joyce. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2: 28-33, 1992). For in vitro evolution, inclusion of recombinant polymerase chain reaction PCR (gene shuffling) offers practical and theoretical advantages over simple recursive point mutagenesis methods. It will rapidly fine tune the mutational load in several parts of the protein by recombining point mutations and wild-type sequences. The technique (and its variations) have been used to enhance enzyme activity, substrate specificity and stability. Gene shuffling is usually achieved by fragmentation of the genes to be shuffled followed by PCR. This method relies on homologous recombination during the PCR reassembly step. Most methods require relatively high levels of sequence similarity between the genes to be shuffled as ‘cross-over points’ appear to occur in these regions.
If sequence similarity is low between the input genes, the majority products tend to be the reassembled parental genes and extensive searches need to be carried out to find recombinants (Kikuchi, M., Ohnishi, K. & Harayama, S. Novel family shuffling methods for the In vitro evolution of enzymes. Gene 236, 159-167, 1999; Ostermeier, M., Shim, J. H. & Benkovic, S. J. A combinatorial approach to hybrid enzymes independent of DNA homology. Nat. Biotechnol. 17, 1205-1209, 1999). Kichuchi et al (1999) have reported on methods for gene shuffling that make use of unique restriction enzyme sites in the sequences of the parental molecules and following cleavage, several PCR steps were carried out to amplify the recombinant genes, a process that allowed hybrid genes to be formed at high frequency.
An entirely different procedure was proposed by Ostermeier et al (1999) that allowed the preparation of combinatorial fusion libraries by progressive truncation of coding sequences of the two parental sequences followed by ligation of the fragments and selection for enzyme activity. Either parent can be used to provide 5′ sequence for the hybrid gene. This procedure, termed iterative truncation for the creation of hybrid enzymes (ITCHY), can accommodate recombination between genes with as little as 50% sequence similarity and was found to give a wider range of crossovers compared with standard gene shuffling techniques.
The present applicant has isolated a gene coding for a thermophilic beta-xylanase that had improved performance in the bleaching of paper pulp. It was desired to investigate the possibility of obtaining mutant derivatives that had enhanced stability and an altered pH optimum. Experiments using error-prone PCR and mis-incorporation mutagenesis followed by gene shuffling allowed the identification of mutant genes that coded for a limited sample of the variations in sequence space but required extensive screening for their identification. Gene shuffling following DNAsel fragmentation of related genes (family shuffling) overwhelmingly gave wild type parental sequences as the major products. After several trials of methods designed to reduce the background, a technique was devised that allows shuffling of genes that differ widely in sequence similarity and G:C content and greatly reduces the appearance of wild type genes. Furthermore, the primer extension conditions may be modified to bias the resulting progeny genes towards any one (or more) of the parental input genes. The present applicant termed this procedure Degenerate Oligonucleotide Gene Shuffling (DOGS) which is described in WO 02/18629.
Although there are many options available to generate mutants, those methods do not necessarily result in useful products or may be too time consuming or unsuccessful. The present inventors have now developed a new method capable of producing potentially many different functional mutants.